Conventionally the L-amino acids have been industrially produced by method of fermentation utilizing strains of microorganisms obtained from natural sources or mutants of the same especially modified to enhance L-amino acid productivity.
There have been disclosed many techniques to enhance L-amino acid productivity, for example, by transformation of microorganism by recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). These techniques are based on the increasing of activities of the enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes from the feedback inhibition by produced L-amino acid (see, for example, Japanese Laid-open application No56-18596 (1981), WO 95/16042 or U.S. Pat. Nos. 5,661,012 and 6,040,160).
On the other hand, the enhancement of amino acid excretion activity may improve the productivity of L-amino acid producing strain. Lysine-producing strain of a bacterium belonging to the genus Corynebacterium having increased expression of L-lysine excretion gene (lysE gene) is disclosed (WO 9723597A2). In addition, genes encoding efflux proteins suitable for secretion of L-cysteine, L-cystine, N-acetylserine or thiazolidine derivatives are also disclosed (U.S. Pat. No. 5,972,663).
At present, several Escherichia coli genes encoding putative membrane proteins enhancing L-amino acid production are disclosed. Additional copies of rhtB gene make a bacterium more resistant to L-homoserine and enhance the production of L-homoserine, L-threonine, L-alanine, L-valine and L-isoleucine (European patent application EP994190A2). Additional copies of the rhtC gene make a bacterium more resistant to L-homoserine and L-threonine and enhance production of L-homoserine, L-threonine and L-leucine (European patent application EP1013765A1). Additional copies of yahN, yeaS, yfiK and yggA genes enhance production of L-glutamic acid, L-lysine, L-threonine, L-alanine, L-histidine, L-proline, L-arginine, L-valine and L-isoleucine (European patent application EP1016710A2).
Earlier the present inventors obtained, with respect to E.coli K-12, a mutant having a mutation, thrR (herein referred to as rhtA23) that is concerned in resistance to high concentrations of threonine or homoserine in a minimal medium (Astaurova, O. B. et al., Appl. Bioch. and Microbiol., 21, 611-616 (1985)). The mutation improved the production of L-threonine (SU Patent No.974817), homoserine and glutamate (Astaurova, O. B. et al., Appl. Bioch. and Microbiol., 27, 556-561, 1991) by the respective E. coli producing strains.
Furthermore, the present inventors have revealed that the rhtA gene exists at 18 min on E.coli chromosome close to the glnHPQ operon that encodes components of the glutamine transport system, and that the rhtA gene is identical to ybiF ORF between pexB and ompX genes. The unit expressing a protein encoded by the ORF has been designated as rhtA (rht: resistance to homoserine and threonine) gene.
Besides, the present inventors have found that the rhtA gene amplification also conferred resistance to homoserine and threonine. The rhtA23 mutation is an A-for-G substitution at position −1 with respect to the ATG start codon (ABSTRACTS of 17th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, Calif. Aug. 24-29, 1997, abstract No. 457). It is known that the nucleotide composition of the spacer between the SD sequence and start codon and especially the sequences immediately upstream of the start codon profoundly affect mRNA translatability. A 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al., Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al., EMBO J., 3, 623-629, 1984). Therefore, it may be predicted that rhtA23 mutation increases expression of rhtA gene.
The rhtA gene encodes a protein that consists of 295 amino acid residues and has calculated molecular weight of 31.3 kDa. The analysis of the RhtA sequence revealed that it is a highly hydrophobic protein containing 10 predicted transmembrane segments. A PSI-BLAST search of the nucleotide sequence of E.coli strain K-12 belonging to the genus Escherichia (Science, 277, 1453-1474 (1997)) revealed at least 10 proteins homologous to RhtA. Among them there are proteins encoded by ydeD and yddG genes. It was shown the ydeD gene is involved into efflux of the cysteine pathway metabolites (Daβler et al., Mol. Microbiol., 36, 1101-1112, 2000; U.S. Pat. No. 5,972,663). The yddG gene has been known as putative CDS, which may encode functionally unknown protein (numbers 3687 to 4568 in the sequence of GenBank accession AE000244 U00096).